There are many veterinary medical conditions, for example hemorrhagic hypotension and anaphylactic shock, in which significant blood loss and/or hypotension (abnormally low blood pressure) occur leading to tissue hypoxia. For subjects with such medical conditions, it is desirable and often critical for their survival to stabilize their blood pressure and to increase the amount of oxygen provided to body tissues by their circulatory systems.
Considerable effort has therefore been expended in developing substances which may be used as resuscitation fluids and/or blood plasma expanders for stabilizing blood pressure and which are capable of carrying and delivering oxygen to bodily tissues. While transfusion of whole blood or commercially prepared red blood cells (packed) may stabilize blood pressure and provide the needed oxygen for survival, there are many costs (blood-typing; testing for antigens/antibodies, viruses and bacteria; processing expense, etc.) and risks (adverse effects on hematology or immunology; pathological organisms, etc.) associated with these therapies. Therefore, the research and development of an alternate oxygen-carrying substance is being pursued in many countries for applications in the transfusion marketplace as well as for novel therapies for many disease conditions.
Hemoglobin, the natural respiratory protein of erythrocytes which carries oxygen to body tissues from the lungs and carbon dioxide from the tissues to the lungs, is a potential alternate oxygen-carrying substance. Erythrocytes contain approximately 34 grams of hemoglobin per 100 ml of cells.
Since native hemoglobin is readily oxidized in air, commercially available preparations may comprise up to 75% methemoglobin, with the balance being primarily oxyhemoglobin (Sigma Chemical Company, St. Louis, Mo., 1996). The high methemoglobin content limits the usefulness of these preparations for use in a blood substitute or organ preservative as well as for use as a diagnostic or biochemical reagent simply because of the cost and labor required to convert or maintain methemoglobin at acceptable minimum concentrations.
Various methods have been developed to isolate hemoglobin from erythrocytes. Bonsen et al., U.S. Pat. No. 4,001,401 (issued Jan. 4, 1977) describes purifying hemoglobin by lysing red blood cells with toluene and then filtering the lysate through diatomaceous earth filters followed by dialysis. Simmonds et al., U.S. Pat. No. 4,401,652 (issued Aug. 30, 1983) and Tye et al., U.S. Pat. No. 4,473,494 (issued Sep. 25, 1984) disclose purification methods involving selective precipitation of hemoglobin with ions followed by ultrafiltration or dialysis. Hsia, U.S. Pat. No. 4,925,574 (issued May 15, 1990) describes purification of hemoglobin by affinity chromatography. Sheffield et al., Biotechnology and Applied Biochemistry, 9, 230-238 (1987) describe the dialysis of red blood cells using a hypotonic solution followed by ultrafiltration through hollow fibers. Finally, Rausch et al, U.S. Pat. No. 5,084,558 (issued Jan. 28, 1992) disclose the use of high performance liquid chromatography to isolate hemoglobin from erythrocytes.
The foregoing methods all have shortcomings in that they are time-consuming and labor-intensive, requiring multiple extraction, dialysis or filtration steps, but most importantly all require a costly postpurification reconcentration step to obtain the resulting hemoglobin solution. Additionally, the foregoing methods eliminate redox enzymes responsible in vivo for maintaining the hemoglobin in a "useful" state. In the absence of these enzymes, hemoglobin is oxidized to methemoglobin which is not capable of binding oxygen. Furthermore, the above inventions do not take advantage of dog red cell physiology which, because of the relative absence of intracellular potassium, allows for quicker and cleaner preparation of a blood substitute because no dialysis step is required to remove a potentially toxic potassium ion. In addition, this dialysis step is required in all prior art and, because dialysis removes numerous small molecular weight compounds, will thereby introduce an alteration of the natural components of the cytoplasmic extract. Another advantage of deriving a canine blood substitute from dog erythrocytes is the homologous basis of the product, i.e., there will be less risk of immunological incompatibility with proteins derived from the same species, especially when these proteins are planned for intravenous infusion in large quantities as part of a blood substitute. Immunological effects of heterologous hemoglobin blood substitutes are a central concern because of the inherent antigenicity of hemoglobin (Yoshioka et al., Biochem. J., 234, 441-447 (1986); Garver et al., Biochem. Genet., 13, 743-757 (1975). Furthermore, the prior art has demonstrated amplified antigenicity with heterologous hemoglobin that has been polymerized or cross linked (Chang et al., Biomat. Artif. Cells Immobilization Biotechnol., 20, 611-618 (1992); Viazova et al., Biull Exsp. Biol. Med., 106, 446-448 (1988); Hertzman et al., Int. J. Artif. Organs, 9, 179-182 (1986). Finally, an adverse side effect of hemoglobin blood substitutes is vasoconstriction or hypertension which may be related to a specific impurity or contaminant of the formulation or may be caused by some unique pharmacology or structure of the hemoglobin in the formulation. Whether the hypertension or vasoconstriction is caused by, or is amplified by, heterologous hemoglobin is as yet uncertain. However, recent studies with a bovine hemoglobin blood substitute in dogs, cats and humans all produced similar vasoconstriction or hypertension (Standl et al., Intensive Care Med., 23, 865-872 (1997); Ulatowski et al., Crit. Care Med., 24, 558-565 (1996); Standl et al., Anaesthetist, 46, 763-770 (1996). In view of the above presentation, it becomes immediately apparent that several needs exist in the development of a canine-derived hemoglobin blood substitute: a method of preparation that poses less risk for contamination either by membrane, membrane-associated cytoskeletal material or by extra processing steps currently used in the art, a method that preserves the endogenous or natural enzymes of the erythrocyte including methemoglobin-reducing substances, a method that, in general, has a large yield with less steps and less cost, a method that produces a canine-derived hemoglobin blood substitute that is immunologically safer with less risk of incompatibility and a method that produces a canine-derived hemoglobin blood substitute that is more efficacious without adverse restriction on blood flow and oxygen delivery that will occur with vasoconstriction or hypertension.